Plant-Based GOS, a new Gold Standard for Special Infant Formulas?

• 184 days ago

Prebiotics are now considered as key ingredients in infant formulas although their use is not mandatory. The demonstration of their multiple benefits on gut health, immunity and global development in infants has led to the development of several prebiotics among which galacto-oligosaccharides (GOS) and human milk oligosaccharides (HMOs) are now considered as gold standards.
While the prevalence of lactose intolerance and cow’s milk allergy are rising in infants and young children, the choice of prebiotic compounds that are safe and suitable for these populations is limited as most prebiotics for infants are produced from dairy raw materials.
A new generation of lactose-free prebiotic substances has emerged with the development of plant-based galacto-oligosaccharides extracted from legumes. Their safety and benefits will be detailed in this article.

Introduction

The gastrointestinal microbiota of breast-fed babies differs from classic standard formula fed infants: While breast milk is rich in prebiotic oligosaccharides, standard infant formula is not (1).

To deliver benefits as close as possible to those provided by breastmilk, infant formulas manufacturers have added prebiotic oligosaccharides to infant formulas since the late 1990s. Prebiotic fibers addition has since become a key differentiating asset for infant and young children formulas manufacturers. From a physiological point-of-view, prebiotic oligosaccharides addition in formulas modulates positively the microbiota of formula-fed infants and makes it closer to that observed in breastfed infants: lower pH, better stool consistency and frequency as well as more balanced composition of the microbiota (such as higher concentration of bifidobacteria) (1).

Galacto-oligosaccharides (GOS) have established as the prebiotic gold standard in infant formulas as they display molecular features similar to structures found in breastmilk. They have shown their ability to shift positively the microbiota and short-chain fatty acids production in formula-fed infants (2, 3, 4) and display a number of potential health benefits such as improvement of stool consistency and frequency (2, 4) and immunity (4, 5).

However all the GOS introduced in the infant nutrition market at the moment are produced from lactose and then cannot be added in formulas targeting infants and young children with lactose intolerance or cow’s milk allergy, i.e plant-based and/or hypoallergenic formulas.

While lactose is currently the main raw material used to obtain GOS, legumes are the major sources of GOS in Human diet (6), even though plant-based GOS have seen limited applications on the nutrition market until now. Recently they have been purified and commercialized for different applications including infant formulas where they are aimed at providing a prebiotic alternative to lactose-based GOS in formulas that require being free of lactose or of any dairy ingredients.

The objective of this work was to evaluate the suitability of plant-based GOS as well as their ability to modulate the gut parameters and microbiota in several preclinical models mimicking the infant gut. To do so, plant-based GOS were compared to other oligosaccharides for their prebiotic potential and their ability to prevent the growth of Clostridium difficile on an in vitro model. Then their suitability and ability to modulate the microbiota in vivo was evaluated in neonatal piglets.

Materials & Methods

In vitro evaluation of the prebiotic potential of plant-based GOS

Design: Fecal samples from infants aged 0-4 months were collected and co-incubated with different oligosaccharides routinely used in infant formulas. Oligosaccharides tested (6 samples per product) were plant-based GOS (P-GOS®), lactose-based GOS (β-GOS), inulin and 2-fucosyl-lactose (2-FL) that were incubated at 4 mg/mL for 24 hours at 37°C.  Additional samples were spiked with Clostridium difficile.

Analyses: Fecal DNA isolation was performed by directly transferring 150 mg of fecal material to a DNA isolation plate where 0.5 mL phenol pH 8.0 was added and samples were mechanically disrupted by bead beating then centrifuged. Aqueous phase was collected and purified for microbiota analysis. Analysis of the microbiota composition was performed by mass sequencing of the V4 hypervariable region of the 16S rRNA gene on an Illumina MiSeq sequencer after amplification of the barcoded DNA fragments spanning the Archaeal and Bacterial V4 hypervariable region.

Statistics: Bilateral Student test with significance level (p) set at 0.05.

Safety and prebiotic potential of plant-based GOS in piglets

Design: 2 groups of 12 pre-weaning farm piglets (Yorkshire Crossbred) were fed a formula without prebiotics (control) or containing plant-based GOS (P-GOS® 8 g/L) for 3 consecutive weeks. During intervention piglets were observed for growth, feed consumption, moribundity, morbidity and abnormal clinical signs. At the end of intervention piglets were sacrificed and blood and tissues were collected.

Analyses: Cecum and colon contents were harvested and pH was measured. Short-chain fatty acids (acetate, propionate and butyrate) in the colon contents were extracted with diethylether and TBDMSi and measured by GC-MS. Lactate was measured with colorimetric method. Bacterial DNA was isolated from fecal sample by using mechanical and chemical lyses ended by a purification step and total number of bacteria, bifidobacteria and lactobacilli were measured with real-time PCR targeting ad hoc regions of the 16S rDNA. The weight of cecum-colon segment was weighted.

Statistics: First a Fisher test was applied to determine variance equivalence of both groups’ values. Second an unilateral Student test was applied to determine differences between groups. Results of the Fisher test were considered to select the adequate Student test to be applied. Significance level (p) was set at 0.05 for all tests performed.

Results

In vitro effect of plant-based GOS on bifidobacteria and Clostridium difficile growth

Plant-based galacto-oligosaccharides increased bifidobacteria levels more than the control, as well as all other oligosaccharides (figure 1A). The bifidogenic effect of plant-based GOS was similar to that observed with β-GOS and superior to other oligosaccharides (2-FL and inulin).

Growth of the 5 most represented Bifidobacteria species was different across conditions (Table 1). At the species level and compared to control, plant-based GOS increased the growth of B. longum, B. pseudocatenulatum and B. gallicum but had an inhibitory effect on B. bifidum. Compared to other oligosaccharides, plant-based GOS promoted increased growth of B. longum compared to 2-FL and β-GOS, increased growth of B. pseudocatenulatum compared to inulin, increased growth of B. breve compared to 2-FL, increased the growth of B. gallicum compared to 2-FL and inulin and decreased B. bifidum growth compared to 2-FL and β-GOS.

When co-incubated with a spike of Clostridium difficile (figure 1B), plant-based GOS, β-GOS and 2-FL elicited a similar growth-inhibition effect on the pathogenic microorganism compared to the control, while inulin displayed a lower inhibitory effect.

       Conditions

Species

Control

2-FL

β-GOS

Inulin

Bifidobacterium longum

0,000 ö

0,000 ö

0,001 ö

0,850 =

Bifidobacterium pseudocatenulatum

0,000 ö

0,305 =

0,000 ø

0,000 ö

Bifidobacterium breve

0,000 ö

0,004 ö

0,060 =

0,004 ø

Bifidobacterium gallicum

0,000 ö

0,004 ö

0,060 =

0,000 ö

Bifidobacterium bifidum

0,000 ø

0,043 ø

0,005 ø

0,902 =

 

 

 

Table 1. Effect of different oligosaccharides co-cultured with infant stools on the 5 major bifidobacterium species detected in samples (p-values calculated vs plant-based GOS values; trend shown with arrows for plant-based GOS group vs comparator)

Figure 1. Effect of different oligosaccharides co-cultured with infant stools during 20 hours on bifidobacteria levels (A) and Clostridium difficile levels (after the medium was spiked with C. difficile at t0) (B)

Effect of plant-based GOS on growth and microbiota in pre-weaning piglets

Body weight, body weight gain, food consumption (Figure 2) were similar at all time points in the Plant-based GOS and Control groups, as well as feed efficiency (16,0 vs 15,1 % respectively ; p>0.05 ; data not shown).

Figure 2. Body weight (dotted line), Body weight gain (bars) and Food consumption (unbroken line) in piglets during intervention ; * p<0,05

The supplementation of formulas with plant-based GOS resulted in lower pH values in the cecum and colon and increased the weight of the cecum-colon segment (Table 2) (13).

 

Table 2. Gut parameters at the end of the piglet intervention (13)

Parameter (unit)

Control

 (Mean±SEM)

Plant-based GOS

 (Mean±SEM)

P-value

Total bacteria

(nb DNA copies/gram)

1,01.1012 ± 8,39.1010

1,34.1012 ± 1,63.1011

0,045

Cecum pH

6,3 ±  0,1

6,0 ±  0,1

0,030

Colon pH

6,7 ±  0,1

6,3 ±  0,1

0,011

Cecum-colon weight (grams)

119,3 ±  7,9

143,6 ± 11,3

0,047

 

Plant-based GOS increased the total count of bacteria in the colon contents (Table 2) as well as bifidobacteria count, while lactobacilli levels were similar to Control (Figure 3A). Levels of acetate and butyrate were increased in the plant-based GOS supplemented piglets while lactate and propionate were not different between groups (Figure 3B).

Figure 3. Bifidobacteria and lactobacilli counts in colonic samples (mean ± SEM) at the end of intervention (A) and Acids levels in colonic samples (mean ± SEM) at the end of intervention

Discussion

Overall our results demonstrate the suitability and nutritional benefits of plant-based GOS in neonates.

The ability to sustain adequate growth is generally considered as a suitability marker when evaluating the interest of new substances introduced in infant formulas (7). In our protocol in neonatal piglets, formula supplemented with plant-based GOS ensured growth patterns similar to those observed with a non-supplemented formula and furthermore improved slightly, despite not significantly, feed efficiency (i.e the ability to extract energy from foods to ensure growth). While rodents are generally the most used animal models to evaluate the safety and suitability of new substances, non-human primates and piglets are considered as more amenable models to mimic infancy conditions as they readily accept infant formulas as nutrient sources (8). Recent position from the European Food Safety Authority (EFSA) clearly supports the use of a repeated dose study in neonatal piglets to evaluate the suitability of new substances, especially when they are not absorbed such as GOS and more generally fibers (9).

The results obtained in piglets support that plant-based GOS display classical features of prebiotic compounds with an acidification of the gut content, the increase in bacterial mass and the increase in the cecum-colon segment weight.

Microbiota results in both experiments support a bifidogenic activity of plant-based GOS similar to β-GOS and superior to inulin and 2-FL. Colonization by bifidobacteria in neonates is considered a key feature of the evolution of the microbiota in early life (10). The genus Bifidobacterium has the ability to process milk oligosaccharides efficiently when milk is the sole source of nutrition. Interestingly while similar bifidogenic effect is observed in plant-based GOS and β-GOS, the specific effect of bifidobacteria shows they each favor specific species, plant-based GOS favoring B. longum and β-GOS favoring B. pseudocatenulatum and B. bifidum, while no difference was observed for B. breve and B. gallicum. The presence of specific bifidobacteria species in early life has been described in a number of epidemiological studies. B. longum, B. bifidum and B. breve are present in both formula-fed and bottle-fed infants in high proportions (11, 12) and have different properties, for example immune-modulating and anti-inflammatory properties for Bifidobacterium breve (13, 14). On the contrary B. pseudocatenulatum is more representative of an adult-type microbiota (15). Interestingly plant-based GOS have limited effects on lactobacilli populations as observed in the piglet trial, while β-GOS which are made of galactose chains with different linkages generally support the growth of lactobacilli (2).

The pattern of short-chain fatty acids production observed with plant-based GOS shows an increased production of acetate and butyrate. While the exact role of SCFAs in early life is not clearly elucidated, existing evidence suggests that they impact beneficially gut maturation processes (16). More specifically acetate has been proposed as a protective agent against enteropathogens (17) and butyrate as an immune-regulating substance increasing the generation of regulatory T cells (18) which are involved in immune reactions linked to allergies and allergens tolerance acquisition (19). Butyrate has also been described as a potential beneficial compound in the management of infant colic (20) and been shown to relieve visceral sensitivity in adults (21).

The inhibitory effect of plant-based GOS on the growth of Clostridium difficile is of particular interest as C. difficile is supposed to partly shape the microbiota of infants and is inversely associated with the presence of B. longum (22). While the impact of C. difficile in the incidence of symptoms such as diarrhea in infants is still debated (23), new evidence suggests that its presence in infancy is linked to allergy development later in childhood (24).

Altogether these findings support the use of plant-based GOS in infant formulas and potential benefits for neonates. While most oligosaccharides currently used in infant formulas are produced from lactose, the plant origin of plant-based GOS makes them suitable in formulas for lactose intolerant and cow’s milk allergic infants.

Contact us for more information

References

(1)  Vandenplas Y, De Greef E, Veereman G. Prebiotics in infant formula. Gut Microbes. 2014;5(6):681-7.
(2) Ben XM, Li J, Feng ZT, Shi SY, Lu YD, Chen R, Zhou XY. Low level of galacto-oligosaccharide in infant formula stimulates growth of intestinal Bifidobacteria and Lactobacilli. World J Gastroenterol. 2008 Nov 14;14(42):6564-8.
(3) Fanaro S, Marten B, Bagna R, Vigi V, Fabris C, Peña-Quintana L, Argüelles F, Scholz-Ahrens KE, Sawatzki G, Zelenka R, Schrezenmeir J, de Vrese M, Bertino E. Galacto-oligosaccharides are bifidogenic and safe at weaning: a double-blind randomized multicenter study. J Pediatr Gastroenterol Nutr. 2009 Jan;48(1):82-8.
(4) Sierra C, Bernal MJ, Blasco J, Martínez R, Dalmau J, Ortuño I, Espín B, Vasallo MI, Gil D, Vidal ML, Infante D, Leis R, Maldonado J, Moreno JM, Román E. Prebiotic effect during the first year of life in healthy infants fed formula containing GOS as the only prebiotic: a multicentre, randomised, double-blind and placebo-controlled trial. Eur J Nutr. 2015 Feb;54(1):89-99.
(5) Osborn DA1, Sinn JK. Prebiotics in infants for prevention of allergy. Cochrane Database Syst Rev. 2013 Mar 28;(3):CD006474
(6) Trinidad TP, Mallillin AC, Loyola AS, Sagum RS, Encabo RR. The potential health benefits of legumes as a good source of dietary fibre. Br J Nutr. 2010 Feb;103(4):569-74
(7) American Academy of Pediatrics (AAP). Clinical testing of infant formulas with respect to nutritional suitability for term infants. Report of AAP Task Force. Elk Grove Village, IL: American Academy, 1988
(8) Institute of Medicine, Food and Nutrition Board, Committee on the Evaluation of the Addition of Ingredients New to Infant Formula. Infant Formula: Evaluating the Safety of New Ingredients. 224 pages. National Academies Press, 2004. ISBN 0309091500, 9780309091503
(9) EFSA Scientific Committee, Hardy A, Benford D, Halldorsson T, Jeger MJ, Knutsen HK, More S, Naegeli H, Noteborn H, Ockleford C, Ricci A, Rychen G, Schlatter JR, Silano V, Solecki R, Turck D, Bresson J-L, Dusemund B, Gundert-Remy U, Kersting M, Lambre C, Penninks A, Tritscher A, Waalkens-Berendsen I, Woutersen R, Arcella D, Court Marques D, Dorne J-L, Kass GEN and Mortensen A, 2017. Guidance on the risk assessment of substances present in food intended for infants below 16 weeks of age. EFSA Journal 2017;15(5):4849, 58 pp.
(10) Arboleya S, Watkins C2, Stanton C, Ross RP. Gut Bifidobacteria Populations in Human Health and Aging. Front Microbiol. 2016 Aug 19;7:1204
(11) Klaassens ES, Boesten RJ, Haarman M, Knol J, Schuren FH, Vaughan EE, de Vos WM. Mixed-species genomic microarray analysis of fecal samples reveals differential transcriptional responses of bifidobacteria in breast- and formula-fed infants. Appl Environ Microbiol. 2009 May;75(9):2668-76
(12) Mevissen-Verhage EA, Marcelis JH, de Vos MN, Harmsen-van Amerongen WC, Verhoef J. Bifidobacterium, Bacteroides, and Clostridium spp. in fecal samples from breast-fed and bottle-fed infants with and without iron supplement. J Clin Microbiol. 1987 Feb;25(2):285-9.
(13) Kruger C, Zhou Y, Thorsrud B A, Morel-Despeisse F, Chappuis E, Safety evaluation of -galactooligosaccharides for use in infant formulas investigated in neonatal piglets, Toxicology Research and Application Volume 1: 1–10

Start a project

We are ready for the challenge
commercial@olygose.com

Find us

Parc Technologique des Rives de l´Oise
60280 Venette - FRANCE

Call us

We are listening to you
+33(0)3 44 90 78 10

© Olygose - Made by 6tematik / AS-im
This website uses some cookies. If you continue navigation, you accept the use of cookies. Warning: blocking cookies prevents the proper operation of the site.